Ó 2009 Wiley-Liss, Inc. Birth Defects Research (Part A) 85:303À313 (2009)
Regulation of Folate Receptor 1 Gene Expression
in the Visceral Endoderm
J. Michael Salbaum,1* Richard H. Finnell,2 and Claudia Kappen1
Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana
Institute for Biosciences and Technology, Center for Environmental and Genetic Medicine, Houston, Texas
Received 31 July 2008; Revised 7 September 2008; Accepted 14 September 2008
BACKGROUND: Nutrient supply to the developing mammalian embryo is a fundamental requirement.
Before completion of the chorioallantoic placenta, the visceral endoderm plays a crucial role in nurturing the
embryo. We have found that visceral endoderm cells express folate receptor 1, a high-afﬁnity receptor for
the essential micronutrient folic acid, suggesting that the visceral endoderm has an important function for
folate transport to the embryo. The mechanisms that direct expression of FOLR1 in the visceral endoderm
are unknown. METHODS: Sequences were tested for transcriptional activation capabilities in the visceral
endoderm utilizing reporter gene assays in a cell model for extraembryonic endoderm in vitro, and in trans-
genic mice in vivo. RESULTS: With F9 embryo carcinoma cells as a model for extraembryonic endoderm, we
demonstrate that the P4 promoter of the human FOLR1 gene is active during differentiation of the cells
towards visceral endoderm. However, transgenic mouse experiments show that promoter sequences alone
are insufﬁcient to elicit reporter gene transcription in vivo. Using sequence conservation as guide to choose
genomic sequences from the human FOLR1 gene locus, we demonstrate that the sequence termed F1CE2
exhibits speciﬁc enhancer activity in F9 cells in vitro, in the visceral endoderm, and later the yolk sac in
transgenic mouse embryos in vivo. We further show that the transcription factor HNF4-alpha can activate
this enhancer sequence. CONCLUSIONS: We have identiﬁed a transcriptional enhancer sequence from the
FOLR1 locus with speciﬁc activity in vitro and in vivo, and suggest that FOLR1 is a target for regulation by
HNF4-alpha. Birth Defects Research (Part A) 85:303–313, 2009. Ó 2009 Wiley-Liss, Inc.
Key words: folate receptor; transcriptional regulation; visceral endoderm; enhancer; HNF4-alpha
INTRODUCTION Shaw et al., 1995), represented a signiﬁcant milestone in
Folate, Birth Defects, and General Health the understanding of congenital malformations. Impor-
tantly, this concept presented a highly feasible therapeu-
Folate deﬁciency has been linked to an increased inci- tic approach to birth defect prevention simply by supply-
dence of congenital malformation (Molloy and Scott, ing vitamin preparations including folate to women who
2001), heightened risk for certain types of cancer (Prinz- wished to become pregnant (Smithells et al., 1981). In
Langenohl et al., 2001; Ryan and Weir, 2001; Courte- fact, periconceptional supplementation with folate (Lock-
manche et al., 2004b), reduced immune system perform- smith and Duff, 1998; Bailey, 2000; Ladipo, 2000) proved
ance (Courtemanche et al., 2004a), anemia, and reduced to be highly beneﬁcial to the conceptus, resulting in sig-
endurance (Lukaski, 2004). Suboptimal folate levels niﬁcantly decreased occurrence of NTDs, craniofacial
appear to be linked to impaired general health (Singh, malformations, and cardiovascular abnormalities among
2004) and neurologic symptoms in aging (D’Anci and newborns (Wald et al., 1991; Gelineau-van Waes and Fin-
Rosenberg, 2004; Kim et al., 2008). Furthermore, our own nell, 2001).
experiments have demonstrated a beneﬁcial effect of fo-
late on the morphogenesis of the skeleton (Kappen et al.,
2004). Historically, the ﬁnding that neural tube defect Presented at the 48th Annual Meeting of the Teratology Society, June 28–July
(NTD) frequencies may be associated with low folate lev- 2, 2008, Monterey, CA.
els in the mother (Smithells et al., 1976; Yates et al., 1987; *Correspondence to: J. Michael Salbaum, 6400 Perkins Road, Baton Rouge,
LA 70808. E-mail: email@example.com
Milunsky et al., 1989), and the resulting general hypothe- Published online 29 January 2009 in Wiley InterScience (www.interscience.
sis that nutritional deﬁciencies could be involved in the wiley.com).
etiology of birth defects (Smithells et al., 1976, 1977; DOI: 10.1002/bdra.20537
Birth Defects Research (Part A): Clinical and Molecular Teratology 85:303À313 (2009)
304 SALBAUM ET AL.
Folate Transport and Cellular Uptake appear that the FOLR1 protein represents the gateway
for this important micronutrient through the visceral
Mammalian cells have developed an elaborate mecha-
endoderm to the embryo itself. It is therefore possible
nism to harvest extracellular folate (Trippett and Bertino,
that the lack of Folr1 in the gene knockout model may
1999), involving extracellular, glycolipid-anchored high-
not only have a cell-autonomous effect on cells of the
afﬁnity folate receptors (Lacey et al., 1989; Wang et al.,
neural tube, but an additional indirect, pleiotropic effect
1996; Wu et al., 1997), a low-afﬁnity transmembrane car-
on the whole embryo. Such pleiotropy may arise from
rier (Moscow et al., 1995; Wong et al., 1995), and a pro-
the absence of Folr1 in the visceral endoderm, a resulting
ton-coupled folate transporter (Qiu et al., 2006). Five fo-
defect in folate transport in the visceral endoderm, failure
late receptor genes have been reported for the human ge-
to supply folate from the visceral endoderm to the
nome (Elwood, 1989; Lacey et al., 1989; Ross et al., 1994;
embryo, and consequently, a condition of folate deﬁ-
Spiegelstein et al., 2000), whereas in the mouse, three fo-
ciency throughout the embryo, with negative consequen-
late receptor genes are present. Expression studies on
ces for cell proliferation and normal morphogenesis.
human folate receptors reveal that FOLR1 is mostly
expressed in epithelial cells (Lacey et al., 1989; Page
et al., 1993; Smith et al., 1999), FOLR3 is speciﬁc for the Functions of the Visceral Endoderm
hematopoietic system (Shen et al., 1994), and FOLR2 The visceral endoderm has important roles in pattern-
(Reddy et al., 1999; Ross et al., 1999; Shen et al., 1994) ing and in nurturing the developing embryo (Brent et al.,
and FOLR4 (Spiegelstein et al., 2000) are found at lower 1990; Bielinska et al., 1999). Crucial patterning signals for
levels in diverse tissues. FOLR4 may play a role for the determination of anterior identity arise from the anterior
immune system because of its expression on regulatory visceral endoderm (Thomas and Beddington, 1996; Tam
T-cells (Walker, 2007; Yamaguchi et al., 2007). The carrier and Behringer, 1997; Beddington and Robertson, 1998),
protein encoded by the RFC1 gene and the proton- whereas visceral endoderm adjacent to extraembryonic
coupled folate transporter PCFT seem to be widely dis- mesoderm is essential for the induction of blood vessel
tributed (Said et al., 1996; Wang et al., 2001; Maddox development (Boucher and Pedersen, 1996; Belaoussoff
et al., 2003; Qiu et al., 2007). Mouse embryos lacking the et al., 1998). In parallel, the visceral endoderm is respon-
Folr1 (folbp1) gene (Piedrahita et al., 1999) are arrested in sible for nutrient uptake and transport to the embryo
their development shortly after gastrulation, fail to close (Cross et al., 1994). This function for nutritional support
the neural tube, and die in utero at mid-gestation, dem- is indispensable during establishment of the chorioallan-
onstrating that the mouse Folr1 gene is essential for em- toic placenta (Brent et al., 1990); once the placenta is
bryonic development. functional, the embryo can switch from histiotrophic to
hemotrophic nutrition (Burton et al., 2001). However, it is
important to consider that crucial developmental proc-
Expression of Folate Receptor Genes during esses, such as neural tube closure and patterning of the
Embryonic Development in the Mouse early heart, occur at a time when the burden of nurturing
the embryo lies with the visceral endoderm (Brent et al.,
The essential nature of folate intuitively would suggest
1990). Thus, it stands to reason that birth defects involv-
that genes involved in folate transport and processing
ing the early embryonic patterning processes in relation
would be ‘housekeeping’ genes, with expression in every
to a lack of nutrients should be interpreted in the context
cell. In contrast, published observations (Saitsu et al.,
of visceral endoderm function. Examples are spina biﬁda
2003) and our own in situ hybridization experiments
and folate deﬁciency (Smithells et al., 1976), or diabetic
(Kappen et al., 2004) demonstrate that this is clearly not
embryopathy and the detrimental nutritional milieu
the case: in the mouse embryo, genes for folate receptors
brought about by maternal diabetes (Reece et al., 1993;
are expressed in distinct and speciﬁc tissue distributions
Reece and Eriksson, 1996). In fact, gene expression
during development. The expression of Folr1 in the neu-
changes in the visceral yolk sac are thought to contribute
ral tube appears to represent a direct link to NTDs
to birth defects in diabetic pregnancies (Reece et al.,
through a cell-autonomous function of the Folr1 gene
2006). Therefore, proper regulation of genes involved in
(Saitsu et al., 2003), and folate supplementation is able to
nutrient uptake and transport in the visceral endoderm is
rescue embryos lacking the Folr1 gene (Spiegelstein et al.,
a crucial prerequisite for successful development of the
2004). However, the literature is not clear about the
embryo itself. A targeted mutation of the transcription
causes for neurulation defects: both neural tube cells
factor HNF4-alpha underscores that view, as many genes
(Copp, 2005) and cells adjacent to the neural tube (Copp
for nutrient transport or metabolism, such as apolipopro-
et al., 1988; van Straaten et al., 1993) are being implicated
teins, glucose transporter 2, transferrin, and cytoplasmic
in a role for NTDs. Thus, a cell-autonomous model for
retinoic acid binding proteins are downregulated in the
Folr1 in NTDs may explain only part of the involvement
visceral endoderm of HNF4-alpha2/2 embryos (Stoffel
of folate in neural tube closure.
and Duncan, 1997). In fact, the failure of HNF4-alpha2/2
In this context, it was interesting to note that the ear-
embryos to complete gastrulation has been ascribed to
liest and strongest expression signal for Folr1 in the
‘‘death by starvation’’ (Copp, 1995; Duncan et al., 1997).
developing embryo occurred in cells of the visceral endo-
derm (Saitsu et al., 2003), and our own data (Fig. 1).
Expression was conspicuously absent from the embryo Regulation of Folate Receptor 1 Gene Expression
itself, raising the question as to how folate enters the ma- Gene transcription is typically dependent on regulatory
jority of cells in the embryo during crucial periods of DNA elements such as promoters and enhancers. Like all
morphogenesis. Regarding the visceral endoderm, we folate receptor genes, the human FOLR1 gene has multi-
found that Folr1 was the only gene of the folate receptor ple promoters that are well characterized. The FOLR1 P1
family expressed in this tissue. Consequently, it would promoter (so designated as the transcript starts at exon 1)
Birth Defects Research (Part A) 85:303À313 (2009)
TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1 305
was studied in KB epidermal carcinoma cells and NIH/ MATERIALS AND METHODS
3T3 ﬁbroblasts (Elwood et al., 1997; Galmozzi et al., In Situ Hybridizations
2001). Both the FOLR1 P1 and the FOLR1 P4 promoter
(transcript starting at exon 4) contain initiator sequen- Decidua with mouse embryos at 7.5 days’ gestation—
ces and respond to the transcription factor SP1 (Sada- with 12 PM on the day of the appearance of a vaginal
sivan et al., 1994; Saikawa et al., 1995). The two pro- plug designated as gestation day 0.5—were dissected
moters exhibit differential activity in KB cells, several from the uterus of FVB mice, embedded in O.C.T. com-
adult tissues, and ovarian cancer cells (Elwood et al., pound (Sakura Finetek, Torrance, CA), frozen, and used
1997; Galmozzi et al., 2001). Furthermore, the transcrip- to generate cryosections of 25 lm thickness. A digoxige-
tion factor vHNF1 activates the P1 promoter in ovarian nin-labeled antisense riboprobe was generated from a
carcinoma cells (Tomassetti et al., 2003), and the FOLR1 mouse Folr1 full-length cDNA clone, and sections were
P4 promoter is modulated by the estrogen receptor in hybridized as described previously (Salbaum, 1998).
cervical and ovarian carcinoma cells (Kelley et al.,
2003). Interestingly, in several cell lines, the FOLR1 Plasmid Constructs
gene appears to regulated in response to cellular Reporter constructs for Luciferase assays were gener-
growth and not in response to folate levels (Doucette ated in pGL3 (Promega, Madison, WI). DNA fragments
and Stevens, 2001), indicating that cellular requirements spanning the P1 and P4 promoter regions, as well as evo-
rather than a simple feedback mechanism control this lutionary conserved sequences ﬂanking the human
gene. A recent study reported genetic variation in the FOLR1 gene, were generated by PCR from commercially
FOLR1 promoter region (Nilsson and Borjel, 2004), with obtained human genomic DNA (Roche, Indianapolis, IN).
potential health implications presumed to be due to Genomic coordinates (UCSC genome browser, human ge-
altered expression of the gene. As is evident from the nome version hg18, March 2006 assembly) for the ampli-
literature on folate receptor gene promoters, most ﬁed fragments were as follows: promoter P1, chr11:
experiments have focused on carcinoma cell lines, with 71,576,404-71,578,395; promoter P4, chr11:71,578,925-
emphasis on FOLR1 gene regulation in cancer. 71,580,883. For conserved sequence elements from the
In contrast, regulatory mechanisms for FOLR1 gene FOLR1 gene, we use the abbreviation F1CE (for Folate re-
expression during embryonic development have not been ceptor 1 Conserved Element) followed by a number;
explored. The expression of FOLR1 in the visceral endo- genomic coordinates were: F1CE1, chr11:71,560,901-
derm is consistent with a role of the visceral endoderm 71,561,809; F1CE2, chr11:71,565,324-71,566,907; F1CE3,
in folate uptake and subsequent release to cells of the chr11:71,591,596-71,592,126. The identity of each ampli-
embryo itself. Therefore, the mechanism that is responsi- ﬁed DNA fragment was conﬁrmed by DNA sequencing.
ble for the speciﬁc expression of Folr1 in the visceral Promoter fragments were generated with ﬂanking MluI
endoderm is fundamental to ensure folate supply to the and XhoI restriction sites for cloning into pGL3
embryo. To identify this mechanism, we have undertaken (Promega); fragments carrying conserved sequences were
reporter gene experiments to gain further insight into the produced with ﬂanking KpnI and MluI sites for cloning
regulatory events that control Folr1 expression during de- upstream of promoter sequences. Deletions in F1CE2-
velopment, with our focus on the visceral endoderm. F1P4-GL3 were generated using existing restriction
Given the importance of folate for prevention of enzyme sites. A reporter plasmid carrying HcRed as re-
human birth defects, it is reasonable to assume that deﬁ- porter gene was generated by replacing the coding
ciencies in folate transport may be cause for susceptibility sequence for luciferase in pGL3 with the HcRed coding
to congenital malformation in humans. Such deﬁciencies sequence from pHcRed-N1.1 (Clontech, Mountain View,
might arise from genetic variation in the structural part CA). The plasmid F1CE2-F1P4-GhcR contains the same
of the FOLR1 gene, but could also be based on mutations assembly of conserved sequence and promoter as F1CE2-
in regulatory regions that are required for proper expres- F1P4-GL3 in the context of the ﬂuorescent reporter, and
sion of FOLR1. Identifying the human regulatory ele- was used for transgenic mouse experiments.
ments for FOLR1 expression would provide a means to
characterize genetic variation in such elements, and
investigate the relationship to birth defect susceptibility. Transfection Experiments
To date, this approach was limited to promoter regions Conditions to grow F9 mouse embryo carcinoma cells
of FOLR1 (Barber et al., 2000), because the existence, as well as differentiation to either visceral or parietal
identity, and location of enhancer sequences were endoderm phenotype were as described elsewhere
unknown. Therefore, rather than using murine sequen- (Braunhut et al., 1992; Dong et al., 1990). Cells were
ces, we chose to attempt identiﬁcation of regulatory seeded in 35-mm dishes at a density of 1 to 2 3 105 cells
sequences from the human FOLR1 locus, using a trans- per dish; transfections using Effectene (Qiagen, Valencia,
genic mouse approach to provide an evolutionary con- CA) and 400 to 600 ng of plasmid DNA were performed
served in vivo context. In this fashion, any human 24 hours after plating. Twenty-four hours after transfec-
sequences with regulatory function in vivo may be read- tion, the transfection mixture was replaced with media
ily checked for potential genetic variation in human pop- containing differentiation agents; for visceral endoderm,
ulations in the future. In this study, we report the identi- cells were treated with 1 lM all-trans retinoic acid
ﬁcation of a sequence from the human FOLR1 locus that (Sigma, St. Louis, MO), whereas for parietal endoderm,
can act as a transcriptional enhancer to direct gene cells were grown on dishes pretreated with 0.1% gelatin
expression speciﬁcally in the visceral endoderm, and we and received 1 lM all-trans retinoic acid and 250 lM
suggest that the FOLR1 gene, like many other genes for dibutyryl-cAMP (Sigma). Sixty hours after induction of
nutrient uptake or transport, is a target for the transcrip- differentiation, a time at which any retinoic acid-medi-
tion factor HNF4-alpha. ated effects of FOLR1 have long ceased, cells were lysed
Birth Defects Research (Part A) 85:303À313 (2009)
306 SALBAUM ET AL.
in GloLysis buffer (Promega), and luciferase activity was
determined using SteadyGlo substrate (Promega). Lucif-
erase values were normalized to the protein content of
the lysate as determined by BCA assay. Each transfection
assay was performed in either ﬁve or ten replicates. For
cotransfections, expression vectors encoding transcription
factors (CMV-HNF4-alpha, CMV-TGIF) were obtained
from the IMAGE Mammalian Gene Collection of full-
length cDNAs (Open Biosystems, Huntsville, AL). The
expression vector pMT7-HNF4-alpha (Jiang et al., 1995)
was kindly provided by Dr. Francis Sladek (University of
California, Irvine, CA). DNA for cotransfection experi-
ments was a mixture of 500 ng of reporter construct
DNA as well as 100 ng DNA of the plasmid encoding a
transcription factor. An expression vector containing the
HcRed ﬂuorescent protein sequence was used as negative
control (in place for transcription factor-expressing plas- Figure 1. Expression of the mouse Folr1 gene in the visceral
mids) for cotransfection experiments and served for nor- endoderm. Sagittal (A) and transverse (B) sections from mouse
malization of reporter activity. For all experiments, fold decidua at 7.5 days’ gestation were hybridized with an antisense
changes were calculated by normalizing all observed val- riboprobe speciﬁc for the murine Folr1 gene. Strong expression
ues to the average of the respective control experiment. was observed in the visceral endoderm, with weaker signal in
Statistical signiﬁcance was determined by performing a the chorion, and no expression detectable in the embryo itself.
double-sided t test on control and experimental values Abbreviations: ave, anterior visceral endoderm; ch, chorion; d,
normalized to the average of the controls. deciduum; ec, ectoderm; h, headfold region; m, mesoderm; ve,
Transgenic Mouse Experiments
The construct F1CE2-F1P4-GhcR was used to generate Activity of FOLR1 Gene Promoters in F9 Cells
transgenic mouse embryos that were then analyzed for Differentiated toward Visceral Endoderm
reporter activity by confocal microscopy. Injection DNA
free of plasmid backbone sequences was generated by We generated reporter constructs comprising 2 kb of
digestion with Asp718 and SalI, followed by agarose gel sequences of either the P1 or the P4 promoter of the
electrophoresis and puriﬁcation (Qiagen). DNA was human FOLR1 gene (Fig. 2A) to test their activity in F9
injected into fertilized oocytes of FVB mice as published embryo carcinoma cells that were differentiated to vis-
(Hogan et al., 1996). At gestation day 7.5 (E7.5) as well as ceral endoderm (Dong et al., 1990; Braunhut et al., 1992).
9.5 (E9.5), embryos were dissected from the uterus of Although we observed no activity from the P1 promoter
CD-1 foster mice, and embryo as well as yolk sac (at in F9 cells under any circumstance, the construct carrying
E9.5) of each specimen were used for imaging HcRed- the P4 promoter showed activity in F9 cells, but only af-
speciﬁc ﬂuorescence on a Zeiss Confocal microscope ter they had undergone differentiation toward a visceral
(Carl Zeiss Inc., Thornwood, NY); all images were taken endoderm phenotype (Fig. 2B). In F9 cells grown without
at identical intensity settings. Genotyping for transgene induction of differentiation, the P4 construct did not ex-
presence was performed on DNA extracted from hibit any activity higher than a promoterless luciferase
embryos after imaging. control vector. We conclude that the P4 promoter of the
human FOLR1 gene has the potential to contribute to
expression of the gene in the visceral endoderm.
Expression of Folr1 in the Visceral Endoderm Activity of FOLR1 Gene Promoters
To reveal sites of expression of the Folr1 gene, we per- in Transgenic Mice
formed in situ hybridization experiments on mouse With the observation of cell type-speciﬁc promoter ac-
embryos at stages prior to neural tube closure. While our tivity from the P4 promoter in the visceral endoderm
results in general conﬁrm previously published data model, we introduced reporter constructs with a b-galac-
(Saitsu et al., 2003), we were intrigued by the high level tosidase reporter gene (Fig. 2A) in transgenic mouse
of expression of Folr1 in the visceral endoderm (Fig. 1) at embryos to test whether promoter sequences of the
embryonic day 7.5, and the yolk sac at later stages of de- human FOLR1, or the mouse Folr1 gene, were sufﬁcient
velopment. The visceral endoderm is a cell layer thought to drive expression of a reporter gene in a pattern resem-
to play an important role for nutrition of the embryo bling the expression of Folr1 in the mouse. Although we
(Brent et al., 1990). We detected only Folr1 expression; were able to generate transgenic specimen at expected
neither Folr2 nor Folr4 expression was found (not shown). frequencies (Table 1), none of those transgenic specimen
It is therefore likely that Folr1 represents the gateway for showed reporter activity that matched expression of
high-afﬁnity folate transport in the visceral endoderm, Folr1. A few embryos transgenic for the human FOLR1
and the regulatory mechanisms that direct expression of P4 construct displayed some lacZ activity, but the spatial
the Folr1 gene in the visceral endoderm are likely to be distributions of these activities were not consistent
of high biologic signiﬁcance for healthy development of between individual transgenic samples, and did not
the embryo. match the known Folr1 expression pattern (Saitsu et al.,
Birth Defects Research (Part A) 85:303À313 (2009)
TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1 307
Figure 2. Activity of the P4 promoter of the human FOLR1 gene. (A) Reporter constructs from the human FOLR1 gene. Promoter con-
structs included the publicly annotated transcription start site as well as 2 kb of upstream DNA for each respective construct. Fireﬂy Lu-
ciferase as well as Escherichia coli b-galactosidase were used as reporter genes. (B) Reporter construct from the mouse Folr1 gene locus.
(C) The human P4 promoter construct shows speciﬁc activity in F9 embryo carcinoma cells only after differentiation towards visceral
2003). We did not observe any reporter activity from sequence F1CE2 to the P4 promoter resulted in an
transgenic samples carrying either the mouse Folr1 P1 or approximately eightfold increase of reporter activity in
the mouse Folr1 P4 construct. We therefore conclude that the visceral endoderm differentiation model, suggesting
the individual P1 or P4 promoter sequences of either the that the F1CE2 sequence can in fact act as an enhancer.
human or the mouse folate receptor 1 gene are not sufﬁ- Deletion of approximately two thirds of the F1CE2
cient to drive gene expression in the correct pattern sequence between the Asp718 and StuI restriction sites
in vivo. (F1CE2-dAS-F1P4) abolished that enhancement, suggest-
ing that this enhancing activity may reside between these
Conserved Sequence Elements at the FOLR1 two coordinates. Deletion of the sequence between the
two PﬂI restriction sites had little effect on enhancer ac-
Gene Locus tivity, as seen for the F1CE2-dP-F1P4 and F1CE2-dPSM-
Because the gene for folate receptor 1 showed a high F1P4 constructs. This suggests that the sequence between
degree of sequence conservation between human and the second PﬂI site and the StuI site, which also contains
mouse, we hypothesized that the regulatory mechanisms a conserved sequence element, is a major contributor to
controlling the expression of the gene might also be con- the observed enhancer effect.
served. We used VISTA to generate a sequence conserva-
tion landscape around the human FOLR1 gene (Fig. 3).
Tissue-Speciﬁc Enhancer Activity of a Conserved
We initially selected the three conserved regions nearest
to the FOLR1 gene and generated reporter constructs Sequence Upstream of the FOLR1 Gene
where each of the conserved sequences were placed in To determine whether the F1CE2 sequence would be
the context of the FOLR1 P4 promoter. An initial transfec- able to confer enhancer activity in vivo, we generated
tion survey experiment with constructs containing a ﬂuo- transgenic mice with a construct carrying the same
rescent reporter (HcRed) suggested that the sequence F1CE2-F1P4 conﬁguration as described in the in vitro
termed F1CE2 conferred transcriptional activation activity experiment, but using a gene for the red ﬂuorescent pro-
upon the P4 promoter. We therefore decided to examine tein HcRed as the reporter gene. We performed transient
the F1CE2 sequence in further detail. transgenic assays where the analysis for reporter activity
was carried out directly on founder embryos. Analysis of
A Conserved Sequence Upstream of the FOLR1 embryos at 7.5 days of gestation (Fig. 5) revealed that all
eight transgenic embryos exhibited red ﬂuorescence re-
Gene Shows Enhancer Activity in vitro
stricted to the region of the visceral endoderm. A second
Using ﬁreﬂy luciferase as the reporter gene, we com- experiment analyzed at 9.5 days of gestation yielded
pared reporter activity for DNA constructs containing the three transgenic specimen; all three showed consistent
FOLR1 P4 promoter in the presence or absence of the red ﬂuorescence in the yolk sac. Fluorescence signals in
F1CE2 sequence, or with parts of the F1CE2 sequence embryos were spurious or not detectable at all, indicating
deleted from the construct. We tested these constructs in that the reporter activity from this construct was speciﬁc
F9 cells differentiated either towards the visceral or the
parietal endoderm model. The results are summarized in
Figure 4. Compared to the promoter-less vector pGL3, Table 1
presence of the P4 promoter resulted in an increase in re- Reporter Constructs in Transgenic Mice
porter activity at about the same magnitude as observed Construct Embryos Transgenic Expression
in the initial experiment; this was observed for both cell
models. Activity of the F1P4-GL3 construct was then hF1P4-LacZ 125 18 0
used to normalize reporter activities and calculate fold mf1P4-LacZ 106 16 0
mf1P1-LacZ 130 10 0
change. We found that addition of the conserved
Birth Defects Research (Part A) 85:303À313 (2009)
308 SALBAUM ET AL.
Figure 3. Conservation proﬁle at the human FOLR1 gene locus. Sequence conservation plot in the vicinity of the human FOLR1 gene
locus. Genes are annotated by arrows, conserved sequence regions used in this study are outlined. Annotation of conservation peaks fol-
lows the VISTA convention, with conserved coding regions colored purple, transcribed non-coding regions in light blue, and conserved
noncoding regions in pink. The colored bar above the conservation landscape indicates the presence of repetitive elements in the human
sequence. Three sequences (termed F1CE, for FOLR1 Conserved Element) with conservation to multiple species were initially chosen to
be included in reporter constructs and tested for transcriptional activation. Both F1CE1 and F1CE2 show deep conservation across verte-
brates, whereas conservation in the F1CE3 sequence is limited to mammals.
for the visceral endoderm at E7.5, and for the yolk sac at
E9.5, but not for the embryo proper. This was in excellent
agreement with our earlier in situ hybridization results
on the expression of the Folr1 gene itself, and suggests
that the human F1CE2 region contains an enhancer
sequence that is sufﬁcient to drive the reporter gene
expression in vivo in a pattern that resembles the expres-
sion of the mouse Folr1 gene. Based on the consistency
between the in vitro and the in vivo results, we conclude
that the F1CE2 sequence can function as an enhancer in
the regulation of folate receptor gene expression in early
Figure 4. Enhancer activity from a DNA fragment upstream of
HNF4-Alpha Can Activate the FOLR1 Enhancer the human FOLR1 gene. F9 embryo carcinoma cells were trans-
fected with various DNA constructs and differentiated either
When we examined the PﬂI-StuI fragment of the towards visceral or towards parietal endoderm. From top: pGL3,
F1CE2 region for conservation and for the presence of basic Luciferase vector without promoter sequences; F1P4-GL3,
potential transcription factor binding sites, we noted a human FOLR1 P4 promoter construct as reference for the experi-
sequence 50 -TGGAATTGGACCT-30 that was identiﬁed by ment; F1CE2-F1P4-GL3, conserved sequence F1CE2 tagged onto
rVISTA software (Loots et al., 2002; Loots and Ovchar- the human FOLR1 P4 promoter construct; F1CE2-dP-F1P4, dele-
tion in the conserved F1CE2 sequence between the two PﬂI
enko, 2004) as a potential binding site for the transcrip- restriction sites; F1CE2-dAS-F1P4, deletion between Asp178 and
tion factor HNF4-alpha. This suggested the possibility StuI sites; F1CE2-dSM-F1P4, deletion between StuI and MluI
that HNF4-alpha might be involved in the function of the sites; F1CE2-dPSM-F1P4, compound deletion with the sequence
F1CE2 enhancer function and thereby contribute to the between the PﬂI sites as well as the sequence between the StuI
regulation of the folate receptor 1 gene. We tested this and MluI sites absent from the F1CE2 sequence. All deletion
possibility by performing cotransfection experiments in constructs share the F1P4-Luciferase portion. Restriction sites
F9 cells differentiated towards the visceral endoderm. For and conservation regions (black) in the F1CE2 sequence are indi-
these experiments, we compared the reporter activity of cated to the right; gray bars represent the sequence present in
the various deletion constructs. The presence of the F1CE2
the F1P4 promoter alone, the F1P4 promoter carrying the
sequence enhances the P4 promoter activity nearly eightfold in
full sequence of the F1CE2 region, and the F1P4 pro- the visceral endoderm paradigm. F1P4 constructs also show ac-
moter with the F1CE2-dPSM deletion. Luciferase reporter tivity in the parietal endoderm model, although the enhancing
plasmids were cotransfected with expression vectors that function of the F1CE2 sequence is diminished. Deletion of the
would express (1) HNF4-alpha from the MT7 promoter sequence between the Asp718 and StuI sites from the F1CE2
(Jiang et al., 1995), (2) HNF4-alpha from the CMV pro- sequence abolished the enhancement of transcription activity.
Birth Defects Research (Part A) 85:303À313 (2009)
TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1 309
Figure 5. Enhancer activity of the F1CE2 sequence in transgenic mice. (A, B, C, D) Images (confocal slices) of four different embryos at
E7.5 that all carry the F1CE2-F1P4-HcRed transgene. Red ﬂuorescence appeared to be restricted to the layer of visceral endoderm (ve)
cells on the outside of the embryo. Stippled white lines show the location of the embryo (e) and the chorion (ch); stippled yellow lines
mark the approximate boundary of the visceral endoderm (ve). (E) Yolk sac (ys) from a transgenic specimen at E9.5 showing bright red
ﬂuorescence (projection view of a stack of confocal images), which is indicative of high reporter activity. (F) Transgenic embryo (slice
view) corresponding to the yolk sac shown in E with very little reporter ﬂuorescence. Stippled area represents forebrain vesicle. No con-
sistent pattern of red ﬂuorescence was detected among independent transgenic embryos. (G) Yolk sac from a second, independent trans-
genic specimen (single confocal slice view), with a strong ﬂuorescence signal. (H) Yolk sac from a nontransgenic specimen without ﬂuo-
rescent reporter activity.
moter, (3) the transcription factor TGIF from the CMV 1999a, 1999b), silencing of the construct might have been
promoter, or (4) the red ﬂuorescent protein HcRed from expected, but was not observed. Surprisingly, the F1CE2-
the CMV promoter. We used CMV/HcRed to control for F1P4 deletion construct carrying the presumed HNF4-
the presence of a very strong enhancer/promoter alpha site, showed signiﬁcantly lower activation in
sequence in the transfected cells, and any sequestering of response to HNF4-alpha than the construct carrying the
general transcription factors that might occur because of intact F1CE2 enhancer element. The construct carrying
the CMV sequence. To test whether any observed effect the F1P4 promoter alone also responded to the presence
would be due to the function of a sequence-speciﬁc DNA of HNF4-alpha, although not nearly as strong as the con-
binding protein and not just due to the increased pres- struct with the F1CE2 enhancer. Therefore, it is likely
ence of any DNA-binding protein, we used the transcrip- that transcriptional activation via HNF4-alpha involves
tion factor TGIF, which is not related to the biologic con- more than a single binding site on F1CE2. Taken to-
text of the experiment. All reporter activities were nor- gether, these results suggest HNF4-alpha as a part of the
malized to the CMV/HcRed co-transfection to calculate regulatory mechanism that controls folate receptor 1 gene
fold-change as a response to presence of HNF4-alpha. In expression speciﬁcally in the visceral endoderm and the
these experiments, we observed that presence of HNF4- yolk sac.
alpha lead to a robust and highly signiﬁcant increase of
reporter activity from the F1CE2-F1P4 construct com- DISCUSSION
pared to control (Fig. 6). The degree of increase was dif-
ferent for the two HNF4-alpha expression plasmids (60- In this study, we demonstrate that a DNA sequence
fold for CMV vs 20-fold for MT7). One reason may be located approximately 13 kb upstream of the P4 pro-
that the two plasmids express different splice variants of moter of the human FOLR1 gene can act as a transcrip-
HNF4-alpha, which are thought to have slightly different tional enhancer for FOLR1 gene expression in the visceral
transcriptional activity (Eeckhoute et al., 2003). More endoderm and the yolk sac. Whereas the P4 promoter
likely though, the difference is due to the higher degree displays activity in an in vitro model of visceral endo-
of expression of HNF4-alpha from the CMV promoter derm, it appears that neither the P4 nor the P1 promoter
plasmid compared with the MT7 promoter plasmid. As of the human or the murine Folr1 genes alone contain the
expected, cotransfection of TGIF as a control for necessary regulatory elements to drive expression of this
increased DNA binding protein content in the cells did gene properly. Addition of the evolutionary conserved
not affect the F1CE2-F1P4 construct in a signiﬁcant way: sequence F1CE2 to P4 reporter constructs confers
as TGIF is a transcriptional co-repressor (Wotton et al., increased transcriptional activity from the construct
Birth Defects Research (Part A) 85:303À313 (2009)
310 SALBAUM ET AL.
tory elements). Therefore, if reporter gene expression
matches between different transgenic founders, it is a
strong indication that the observed reporter gene expres-
sion is due to a biologic function on the transgene
sequence, and not due to the genomic integration site.
The fact that we observed excellent congruency of re-
porter expression at both developmental time points is a
compelling argument that the F1CE2 sequence harbors
transcriptional enhancer function. The full characteriza-
tion of the F1CE2 enhancer (e.g., the developmental time
course) will have to await the establishment of transgenic
Our experiments show that the F1CE2 sequence has in-
structive properties and is sufﬁcient to drive expression
in the visceral endoderm. However, we used only the P4
promoter of FOLR1 in the pertinent experiments. We can-
not rule out that the F1CE2 enhancer could also activate
the P1 promoter of FOLR1. Preliminary experiments (not
shown) using a heterologous promoter from the ICP4
Figure 6. HNF4alpha can activate the F1CE2 sequence from the gene of herpes simplex virus indicate that the F1CE2
FOLR1 gene. Cotransfection experiments indicate that the con- sequence can activate such a heterologous promoter at
struct carrying the entire F1CE2 enhancer sequence responds least in F9 cells differentiated to visceral endoderm, and
very strongly to the presence of HNF4-alpha. F1P4-GL3, human
FOLR1 P4 promoter fused to a luciferase reporter; F1CE2-F1P4-
thereby fulﬁll the classic deﬁnition of an enhancer. It
GL3, the F1P4-GL3 construct carrying the entire F1CE2 enhancer therefore stands to reason that the P1 promoter of the
sequence; F1CE2-dPSM-F1P4-GL3, the F1P4-GL3 construct with FOLR1 gene may also be activated by the F1CE2
a deletion version of the F1CE2 enhancer. A plasmid expression sequence, although this remains to be proven experimen-
HcRed (instead of any transcription factor) from the CMV pro- tally.
moter was used as control. Values were normalized to the aver- Although our data demonstrate the biologic activity of
age of the control experiment to determine fold-changes. F1CE2, our studies cannot address whether the F1CE2
Cotransfection of HNF4-alpha leads to a strong activation of the sequence is solely responsible, or even necessary for vis-
reporter construct carrying the entire F1CE2 sequence, with only ceral endoderm expression of FOLR1. In fact, the DNA
mild increases seen for the F1P4 promoter alone, or for the con-
struct with the deletion version of the F1CE2 sequence.
sequence conservation at the FOLR1 gene locus would
suggest that there may be other sequences in the vicinity
of the FOLR1 gene that may have similar properties as
F1CE2. Closer examination of the F1CE2 sequence
revealed that F1CE2 is in fact a remnant of a folate recep-
in vitro, and allows tissue-speciﬁc expression congruent tor gene, or a folate receptor pseudogene. No transcripts
with Folr1 gene expression in transgenic experiments have been reported to arise from the human F1CE2
in vivo. These results are consistent with our interpreta- sequence, but a close sequence relationship for three
tion that the F1CE2 sequence functions as an enhancer. small subregions on F1CE2 to the last three coding exons
In this context, it is important to note that in the ab- of other folate receptor genes is readily recognizable (Fig.
sence of developmental expression data for the human 7). The F1CE2 sequence has a positional match in primate
FOLR1 gene, we are using the mouse Folr1 gene and its genomes, as well as in the genomes of dog and horse: in
expression as a model and as guidance to evaluate the these genomes, a sequence matching F1CE2 exists in a
activity of sequences from the human FOLR1 gene locus. location upstream of the cognate folate receptor 1 gene.
The fact that the human DNA sequences used in this Interestingly, no such positional match exists between the
study were able to generate speciﬁc transcriptional
responses in our in vitro model of murine origin, as well
as in transgenic mouse experiments in vivo, would argue
that regulatory mechanisms are conserved between the
human and mouse version of the folate receptor 1 gene.
This would suggest that expression of the human gene
may occur in a manner similar to the mouse gene during
We used a strategy of analyzing the transgenic speci-
men directly; in this fashion, every reporter expression
signal arose from an independent transgenic event. Since
transgene DNA introduced by pronuclear injection typi- Figure 7. Conservation of the F1CE2 sequence. Comparison of
cally integrates in a random manner, it is highly unlikely the F1CE2 sequence to human and mouse genomes revealed the
presence of three conserved regions (dark shading). The match
that two independent transgenic events occur at the same
to the F1CE2 sequence itself in the human genome is not shown.
genomic integration site. Consequently, the genomic Capitalized gene names are human genes, u-F1CE2 denotes a
neighborhood is unique for each transgenic event. The sequence upstream of the F1CE2 sequence in the human genome
genomic neighborhood of a transgene can exert strong that is also a remnant of a folate receptor gene. Percentage of
inﬂuences on a transgene expression (e.g., through meth- sequence identity over a given nucleotide span is indicated. The
ylation patterns or through the presence of strong regula- regions of 525bp and 177bp are not drawn to scale.
Birth Defects Research (Part A) 85:303À313 (2009)
TRANSCRIPTIONAL ENHANCER FOR FOLATE RECEPTOR 1 311
human and mouse genomes. In the mouse, the only mice, or the removal of potential HNF4-alpha binding
sequences with relationship to F1CE2 are in fact the sites from the F1CE2 enhancer sequence. Given that there
sequences for the two folate receptor genes Folr1 and are other conserved potential transcription factor binding
Folr2. In line with the hypothesis of conservation of sites on the F1CE2 sequence, it is likely that F1CE2
expression and regulation, we propose that sequences enhancer function requires other factors besides HNF4-
controlling the murine Folr1 gene may reside in either alpha. Nevertheless, our results raise the possibility that
the Folr1 gene itself, or in the neighboring Folr2 gene. folate transport is in fact a process controlled by HNF4-
Based on sequence similarity and on the presence of alpha, and support the concept that HNF4-alpha func-
another potential HNF4-alpha binding site, it appears tions to integrate the general process of nurturing the
that the Folr2 sequence is the more likely candidate for embryo before the completion of the placenta and the
harboring enhancer function. It is therefore reasonable to onset of hemotrophic nutrition.
assume that the human folate receptor gene locus on This study provides a ﬁrst insight into the mechanisms
chromosome 11 with it’s higher complexity of folate re- that regulate FOLR1 gene expression during critical times
ceptor genes – besides FOLR1 and FOLR2, there is also of embryonic development. Understanding the signals
the FOLR3 gene, as well as two FOLR gene remnants that impinge on these mechanisms and their relationship
(one of which is F1CE2) – might harbor more than one to maternal folate status will provide further insight into
sequence capable of driving FOLR1 expression in the vis- the regulation of FOLR1 gene expression and folate trans-
ceral endoderm. port in the embryo.
It is well known that folate supplementation can
reduce the incidence of NTDs (Smithells et al., 1981). In
regard to the timing of neural tube closure, it is impor- ACKNOWLEDGMENTS
tant to note that the process of neurulation occurs at a Part of this work was carried out at and funded by the
time when the chorioallantoic placenta has not been fully Munroe-Meyer Institute at the University of Nebraska
established. At that time, the visceral endoderm has the Medical Center. We wish to acknowledge technical help
function of supplying nutrients to the embryo. In the by Andrew Wall, Ryan Taylor, and Don Harms, help
quest of understanding how folate supplementation can with confocal microscopy by Dr. Bernd Fritzsch and
exert its beneﬁts on the embryo, it appears that the func- Heather Thomas, as well support by grants NIH
tion of the visceral endoderm is of high biologic signiﬁ- DE016315 (to RHF) and NIH DK063336 (to CK).
cance. In the mouse, the visceral endoderm mediates
histiotrophic nutrition of the embryo, and it stands to
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